Species‐ and sex‐dependent changes in body size between 1892 and 2017, and recent biochemical signatures in rural and urban populations of two ground beetle species

Abstract Increasing urbanisation and intensified agriculture lead to rapid transitions of ecosystems. Species that persist throughout rapid transitions may respond to environmental changes across space and/or time, for instance by altering morphological and/or biochemical traits. We used natural history museum specimens, covering the Anthropocene epoch, to obtain long‐term data combined with recent samples. We tested whether rural and urban populations of two ground beetle species, Harpalus affinis and H. rufipes, exhibit spatio‐temporal intraspecific differences in body size. On a spatial scale, we tested signatures of nitrogen and carbon stable isotopes enrichments in different tissues and body components in recent populations of both species from urban and agricultural habitats. For body size examinations, we used beetles, collected from the early 20th century until 2017 in the Berlin‐Brandenburg region, Germany, where urbanisation and agriculture have intensified throughout the last century. For stable isotope examinations, we used recent beetles from urban and agricultural habitats. Our results revealed no spatio‐temporal changes in body size in both species' females. Body size of H. rufipes males decreased in the city but remained constant in rural areas over time. We discuss our findings with respect to habitat quality, urban heat and interspecific differences in activity pattern. Although nitrogen isotope ratios were mostly higher in specimens from agricultural habitats, some urban beetles reached equal enrichments. Carbon signatures of both species did not differ between habitats, detecting no differences in energy sources. Our results indicate that increasing urbanisation and intensified agriculture are influencing species' morphology and/or biochemistry. However, changes may be species‐ and sex‐specific.

In urban areas, exhaust gases from industry and traffic produce pollution (Fenger, 1999), artificial night lighting leads to an imbalance between day and night (Falchi, 2019), and construction works conduce fragmentation and loss of habitats (Liu et al., 2016). The increasing proportion of artificial surfaces, for example for buildings and roads, is influencing microclimatic conditions, resulting in increasing temperatures of 1-5°C in cities compared to undeveloped surrounding areas (Kalnay & Cai, 2003). Such urban heat islands (Oke, 1973;Rizwan et al., 2008) will be more accentuated by global warming (Alcoforado & Andrade, 2008), resulting in more frequent heat waves in cities (Fenner et al., 2018). In agricultural landscapes, however, monocultures lead to habitat homogenisation (Jongman, 2002), the use of herbicides and/or insecticide influence biodiversity negatively (Geiger et al., 2010) and the application of chemical and/or biological fertilisers result into increased nitrogen enrichments, as well as other nutrients, to the environment (Freyer & Aly, 1974;Jenkinson, 2001;Shearer et al., 1974;Vitousek et al., 1997;Zarzycki & Kopeć, 2020). These environmental changes can cause to rapid transitions to altered environments over time (Hobbs et al., 2013;Jeltsch et al., 2013), influencing species composition and function, giving rise to anthropogenically impacted ecosystems (Harris et al., 2006;Ricciardi, 2007;Root & Schneider, 2006). Consequently, many native species disappear from anthropogenically impacted ecosystems, whereas non-native species establish via human transport or range extension (Kenis et al., 2009;Ziegler, 2011). However, some native species persist through the transition process (Doudna & Danielson, 2015;Van't Hof et al., 2011), because they are able to cope with or adapt to new conditions (Cook & Saccheri, 2013;Giraudeau et al., 2014).
In the Berlin-Brandenburg region, Germany, rapid transitions to anthropogenically impacted environments took place across time, resulting in a densely populated metropolis of Berlin, surrounded by the rural federal state of Brandenburg that is mainly covered by agricultural monocultures (Cochrane & Jonas, 1999). For answering the question on how native species manage to persist through such rapid transitions to anthropogenically impacted environments, natural history museum collections can provide a source of such persisting species, covering time series of one region. For this purpose, morphological traits of museum voucher specimens can be used as proxies for species' ecological and physiological modifications over time (Keinath et al., 2020(Keinath et al., , 2021Niemeier et al., 2020;Rocha et al., 2014).
Body size is a useful morphological trait, indicating habitat quality (Blake et al., 1994;Chungu et al., 2018;Kotze & O'Hara, 2003;Lövei & Magura, 2022;Magura et al., 2020;Niemelä et al., 2002), and correlates with many life-history aspects, such as reproduction rate, dispersal ability or developmental time (Peters, 1983). Gray's increasing disturbance hypothesis predicts that mean body size should decrease from less to more disturbed habitats, in the present context from rural to urban areas (Gray, 1989).
Especially ectothermic animals, such as insects, may be impacted by urbanisation because their development depends on environmental temperature, and thus might be sensitive to changing microclimatic conditions and anthropogenically modified habitats in cities. Is the food source of an insect larva limited, higher temperatures lead to faster larval development and an earlier start of metamorphosis, resulting in smaller-sized imagines (Gillooly et al., 2001;Kingsolver & Huey, 2008). In holometabolic insects, this mechanism is driven by the timing of key hormonal events involved in moulting and metamorphosis (Davidowitz et al., 2003(Davidowitz et al., , 2004. Thus, living in the city may result in smaller-sized insects in comparison with insects occurring in cooler rural surroundings (Brans et al., 2017;Kotze et al., 2011) when rural habitat quality is more suitable than the urban one (Gillooly et al., 2001;Kingsolver & Huey, 2008; Figure 1).
Apart from morphological traits stable isotope composition reflects the habitat conditions of an individual (Peterson & Fry, 1987;Tieszen & Boutton, 1988). In organisms, carbon and nitrogen are enriched with two stable isotopes, 13 C and 12 C, and 15 N and 14 N, respectively (Rosing et al., 1998). The stable carbon isotope signatures provide information about the respective energy base of an animal's diet (Gratton & Forbes, 2006;Ponsard & Arditi, 2000), reflecting the relative proportion of C3 and C4 plants in its respective environment (Degens, 1969;Schwarcz, 1969). The nitrogen isotope composition provides information about the position of an individual in the food web (Birkhofer et al., 2011), and whether the individual lived in a nitrogen rich environment, such as in an agricultural landscape with intense application of chemical and/or biological fertilisers (Freyer & Aly, 1974;Jenkinson, 2001;Shearer et al., 1974;Vitousek et al., 1997). Thus, populations showing different 15 N/ 14 N ratios may indicate different habitat conditions in which the populations are occurring, such as in intensively managed agricultural and less impacted habitats (Birkhofer et al., 2011).
The composition of stable isotopes may also differ in different tissues and/or body components, depending on its respective turnover time. Consequently, it is possible to determine long-term as well as short-term dietary preferences of one individual specimen. Chitin has slow turnover and because holometabolic insects are not moulting after metamorphosis, its isotopic signature reflects the nutritive status of an individuals' larval stage. However, tissues with fast turnover, like muscles, reflect the dietary conditions of an adult individual (Gratton & Forbes, 2006;Peterson & Fry, 1987).
Thus, analyses of stable isotopes from different tissues and/or body components may provide information about whether different development stages of an individual exploit nutrient sources from different habitats that differ in their nitrogen or carbon enrichments. Different stable isotope enrichments may thus indicate that an individual dispersed from one into another habitat (Hood-Nowotny & Knols, 2007;Schallhart et al., 2009).
Among holometabolic insects, carabid beetles are a suitable and well-known indicator group of human-caused disturbances and habitat quality, such as urbanisation and agricultural land use.
Ground beetles respond differently to urbanisation (Magura et al., 2020Sadler et al., 2006;Sukhodolskaya & Saveliev, 2014). On a community level, Blake et al. (1994) and Ribera et al. (2001) showed that smaller ground beetle species are more abundant in highly disturbed areas than larger ones, whereas Magura et al. (2020) mainly found smaller species of silvicolous ground beetles in urban areas, due to their higher sensitiveness to habitat fragmentation and higher environmental temperatures. On an intraspecific level, the studies by Weller and Ganzhorn (2003) and Weller (2000) showed decreasing body size in the generalist ground beetle species Carabus nemoralis (Müller, 1764) towards the city centre.
For our study, we selected two ground beetle species, Harpalus affinis (Schrank, 1781) and H. rufipes (De Geer, 1774). Both species are common and well-represented in the collection of the Museum für Naturkunde, Berlin, comprising specimens from Berlin and Brandenburg that cover the past 125 years and are easy to collect in recent times in order to extend time series and for stable isotope examinations. Both species occur in little impacted environments and in open, altered habitats such as meadows, pastures, arable land and urban fallows and parks (Anjum-Zubair et al., 2015;Deichsel, 2006;Harrison & Gallandt, 2012;Sunderland et al., 1995;Townsend, 1992). Although both species are generalists in adult and larvae stages, they predominantly feed on weed seeds (Bažok et al., 2007;Luff, 2002;Sunderland et al., 1995;Toft & Bilde, 2002;Townsend, 1992;Trautner, 2017).
The first aim of our study is to examine changes in body sizes in urban and rural populations of Harpalus affinis and H. rufipes across the last 125 years in the Berlin-Brandenburg area, Germany.
Reasoned by both beetles' predominantly food source, weed seeds, we predicted urban habitats to be lower in habitat quality than rural landscapes with high amount of agriculture. With additionally higher temperatures in urban areas, we assumed specimens of both species to be smaller when occurring in urban areas, whereas we assumed F I G U R E 1 Predicted anthropogenic influence on body size of ground beetles, in space and time. Along a land-use gradient we expected to find smaller-sized beetles in urban habitats compared to agricultural habitats (a), in rural areas (composed of near-natural and agricultural habitats) we expected body sizes to change to a lesser extent (b), whereas we expected beetles to shrink in the city over time (c). body size in specimens of both species occurring in rural areas to exhibit overall larger body size. We further predicted both species to be smaller in recent times than in the past, due to increasing habitat destructions and temperatures in the city of Berlin over time, whereas we assumed both species' body size to decrease to a lesser degree in our rural study region over time reasoned by higher habitat quality for both species and lower temperature increase (Brans et al., 2017;Kotze et al., 2011;Li et al., 2018; Figure 1). Additionally, we anticipate higher variability in body size in both species when occurring in the city, due to higher habitat heterogeneity in urban areas (such as parks, ruderal areas, private gardens, green strips) than in rural landscapes (Falk, 1976;Gill & Bonnett, 1973;Tischler, 1973).
Second, we aim to examine stable nitrogen and carbon isotope enrichments in different tissues and body components of both study species in recent populations from intensely managed agricultural habitats and urban habitats. We predicted higher nitrogen enrichment in beetles occurring in agricultural habitats, compared to urban ones, reflecting the use of fertilisers as shown in Birkhofer et al. (2011) for agricultural arthropods. As a consequence of different plant assemblages, we assumed stable carbon isotope composition to differ between beetles originating from agricultural versus urban habitats (Ponsard & Arditi, 2000). Finally, we expected intraspecific differences of isotopic signatures in different tissues and/or body components (Gratton & Forbes, 2006;Peterson & Fry, 1987), if adult beetles dispersed into habitats that differ in nitrogen enrichments to those of the larval stage (Hood-Nowotny & Knols, 2007;Schallhart et al., 2009).

| Study area
In the Berlin-Brandenburg region of Germany, rapid environmental transitions occurred especially during the last century (Cochrane & Jonas, 1999). The city of Berlin is a fast-growing metropolis with increasingly high level of urbanisation and an increasing human population (Antrop, 2000). Brandenburg, the German federal state surrounding Berlin, mostly consists of rural areas comprising nearnatural environments, as well as intensively managed agricultural monocultures (Cochrane & Jonas, 1999; Figure 2). In 2017, Berlin comprised 4108 inhabitants/km 2 , whereas only 84 inhabitants/km 2 lived in Brandenburg (Statistik Berlin Brandenburg, 2007). Li et al. (2018) measured higher temperatures in Berlin compared to its rural surroundings, that is 5.38 and 2.84 K between day and night-time, respectively. By using long-term data (1893-2017), Fenner et al. (2018) found a significant temperature increase in Berlin, including increasing frequencies of summer heat waves.
All beetles were classified by species, sex and origin. For body size analyses, beetles from Berlin were classified as originating from 'city area', Brandenburg specimens were classified as originating from 'rural area'. Beetles from recent fieldwork and museum vouchers with detailed habitat information were used for in depth habitat TA B L E 1 Numbers of examined specimens of Harpalus rufipes and H. affinis per sex, city and rural areas, time period (1 = 1892-1949; 2 = 1957-1998; 3 = 2016-2017) and urban and agricultural habitats that could be clearly assigned from museum specimens labels or from recent fieldwork.
Numb. Of specimens comparisons between 'urban habitats' and 'agricultural habitats'.
Therefore, we classified beetles from 'city areas' into 'urban habitats' when sampled in ruderal sites, dumpsites, edges of streets and parks, and beetles from 'rural areas' into 'agricultural habitats' when sampled in agricultural landscapes (see numbers of examined beetles per classification in Table 1). We suspect that museum vouchers of both species without any further sampling information except 'Berlin', collected in the 19th century, were sampled at the periphery of Berlin. However, we assigned them to the category 'city area' because the periphery of Berlin, even at that time, can be already regarded as urbanised in comparison with its rural surroundings (Reif, 2002).
For temporal comparisons, we included beetles classified as originating from 'city area' and 'rural area'. Beetles of each category were assigned to one of three distinct time periods: 1892-1949, 1957-1998 and 2016-2017. Time period classifications were based on a combination of practical reasons (availability of sufficient numbers of beetles) and the history of urbanisation and agricultural techniques in our study region (see Keinath et al., 2020).
During the first time period , the Berlin-Brandenburg region was mainly impacted by the effects of the industrial revolution and First and Second World Wars (Ribbe et al., 2002a(Ribbe et al., , 2002b. Berlin expanded to 'Groß-Berlin' in the 1920s, resulting in rapid construction of buildings and streets (Buesch & Haus, 1987). Then, the Berlin population was larger than ever (Ribbe et al., 2002b).
Industrialisation led to high level of environmental pollution (Pamme, 2003). During the second time period , the reconstruction of Berlin was finalised. Berlins' human population was, however, much below the pre-war conditions (Ribbe et al., 2002b;Schildt & Sywotlek, 1993). By the end of the 1950s, the first environmental protection measures were implemented, including efforts to reduce environmental pollution in the city (Pamme, 2003;UBA, 1998;UNEP/WHO, 1993). Therefore, we treated this time  (Pamme, 2003). Whereas numerous museum specimens were available from the first two time periods, only a few specimens were available from the most recent period. We therefore complemented the museum specimens by additional sampling (see all data https://doi.org/10.7479/stwb-nf68). H. rufipes: ±0.09 mm) were determined by the mean accuracy of repeated measurements of a randomly chosen subset of 10 specimens per species. All measurements were taken by one person without prior knowledge of sex, region of origin and habitat of the respective specimens.

| Stable nitrogen and carbon isotopic composition
For stable nitrogen and carbon isotope analyses, we selected 20 recently collected specimens of each species. We examined 10 specimens (5 males and 5 females) of H. affinis from nine dry grass- Tables 2 and A1 in Appendix 1). Neither species in both sexes differed in their body size between habitats (agricultural vs. urban) nor between areas (rural vs. city; Tables 2 and A1; Figures 3 and 4a,b).
Likewise, body size in females in both species remained constant in rural and city areas over time (Tables 2 and A1; Figures 3c and 4c).
This was also the case for male H. affinis (Tables 2 and A1; Figure 3d) from rural habitats over time.
In H. affinis, no spatial differences in size variability were found (Tables 2 and A2)

| Stable isotopes
The stable nitrogen and carbon isotope composition within particular tissues and body components of both species did not differ between sexes (Tables 3 and A3). For subsequent analyses, we thus   Figure 5a).
We detected no significant differences in carbon isotope signature values or value variability in tissues/body components of H.
affinis and H. rufipes related to different habitats (Tables 3 and A3; Figure 5c,d). The comparison of nitrogen and carbon stable isotope signatures between cuticula, legs and muscles of specimens of both species either from agricultural or from urban habitats yielded no significant differences either (Table A4).

F I G U R E 3 Body size of Harpalus affinis from agricultural and urban habitats (a) and rural and city areas (b) of females (rose boxes) and males (blue boxes)
; females (c) and males (d) in rural (green boxes) and city (grey boxes) areas over time. Boxes mark the interquartile ranges, whiskers indicate the minimum and maximum values, horizontal lines in boxes indicate medians, dots above and below boxes represent outliers, numbers below boxes indicate sample sizes, dotted brackets with stars indicate significant differences in variability of body size (*p < .05).

| DISCUSS ION
Intraspecific morphological and biochemical trait changes, caused

F I G U R E 4 Body size of Harpalus rufipes from agricultural and urban habitats (a) and rural and city areas (b) of females (rose boxes) and males (blue boxes); females (c) and males (d) in rural (green boxes) and city (grey boxes) areas over time.
Boxes mark the interquartile ranges, whiskers indicate the minimum and maximum values, horizontal lines in boxes indicate medians, dots above and below boxes represent outliers, numbers below boxes indicate sample sizes, dotted brackets with stars indicate significant differences in variability of body size, drawn through brackets with stars indicate significant differences in body size (*p < .05; **p < .01). (Fenner et al., 2018;Gillooly et al., 2001;Kingsolver & Huey, 2008;Li et al., 2018). Across a spatial gradient from rural to urban habitats, we assumed stable nitrogen and carbon enrichments to be reflected in specimens' tissues and body compounds when occurring in differently human-induced habitats. We detected some intraspecific morphological trait changes to occur across 125 years, depending on the respective species, activity pattern and sexes, as well as biochemical trait changes between populations occurring in differently human impacted habitats.

| Body size in carabids over time
We found no spatio-temporal changes in body size in both sexes of  (1892-1949; 1957-1998).
In many taxa, intraspecific variation in body size is known to be influenced by environmental temperatures, primarily in ectotherms, that is amphibians (Reading, 2007), fish (Todd et al., 2008) and insects (Kingsolver & Huey, 2008). In insects, occurring in habitats with limited food sources, higher environmental temperatures lead to an earlier start of metamorphosis, resulting in smaller-sized imagines (Gillooly et al., 2001;Kingsolver & Huey, 2008). Because the predominant food source in both studied ground beetle species are weed seeds (Bažok et al., 2007;Luff, 2002;Sunderland et al., 1995;Toft & Bilde, 2002;Townsend, 1992;Trautner, 2017), we assumed urban habitats, in addition to their higher temperatures, to be lower in habitat quality than rural areas with a high amount of agricultural landscapes.
Berlin exhibits higher temperatures than its rural surroundings (Li et al., 2018), and its temperature increased with increasing urbanisation over time (Fenner et al., 2018;Tseng et al., 2018). Thus, higher and/or increasing temperatures within the city were assumed to result in smaller and/or decreasing body size in both species, as described for other arthropods (Brans et al., 2017;Kotze et al., 2011). size is more constrained than males, based on ability to produce eggs and/or behavioural differences (Thornhill & Alcock, 1983). In addition, we did not detect any spatial intraspecific differences in body size between habitats (agricultural, urban) and areas (rural, city) in both species. Thus, we refute our hypotheses that insufficient habitat quality in the city and increasing urban heat lead to smaller-sized beetles compared to rural areas in space and time.
The reason for species-and sex-specific decrease in body size over time might be caused by other urban environmental conditions that changed over time. Therefore, differences in the biology between H. affinis and H. rufipes, as well as between H. rufipes sexes have to be considered. Both species differ in their activity pattern.
Whereas H. affinis is diurnal, H. rufipes is predominantly nocturnal (Wrase, 2004). their reproductive energy in mating effort, that is movement to encounter mates, while females expend more energy in egg production (Thornhill & Alcock, 1983).
Because of H. rufipes' nocturnal activity, this species is known to be attracted to artificial nightlights (Kegel, 1990;Matalin, 2003;Szentkirályi et al., 2003). Potentially, male H. rufipes, searching for F I G U R E 5 Stable δ 15 N and δ 13 C (in ‰) in cuticula, legs, and muscles of Harpalus rufipes (a; c) and H. affinis (b; d) from agricultural (yellow boxes) and urban (grey boxes) habitats, boxes mark the interquartile ranges, whiskers indicate the minimum and maximum values, horizontal lines in boxes indicate medians, dots above and below boxes represent outliers, numbers below box plots provide sample sizes, black brackets with stars indicate significant difference between stable isotope values in tissues/body components between habitats, dotted bracket with stars indicate significant differences in variability of stable isotope values in tissues between habitats (*p < .05; **p < .01).
females in the city nocturnally, might be distracted and attracted by streetlights, captured in its light beam, unable to escape and die due to predation or exhaustion, as was shown for other nocturnal insects (Eisenbeis, 2006;Manfrin et al., 2018;Rydell, 1992).
A study by Lagisz (2008) showed that relatively larger wing size in Pterostichus oblongopunctatus (Fabricius, 1787) is related to better dispersal abilities. Matalin (2003) examined in different ground beetle species, including H. rufipes, relative wing surface to be an indicator for dispersal abilities. Thus, larger specimens might have better dispersal abilities than smaller ones. Hence, larger H. rufipes males might have better flight capacities to disperse over longer distances when searching for females and might therefore be more disturbed by street lights than smaller ones. Although larger individuals are better dispersers, this may come with the cost of lower reproductive rates (Crawley, 1989). Thus, increasing the number of streetlights in Berlin across the past 150 years (Eisenbeis & Hänel, 2009;Keinath et al., 2021;Kyba et al., 2017) might have led to higher selection pressures on large-sized males and resulted in an adaptive advantage for smaller-sized males in the city.
By contrast, body sizes of H. rufipes males remained nearly constant in rural areas over the investigated timeframe. Rural environments are less impacted by street lights (Rich & Longcore, 2006) and should have little impact on H. rufipes males. However, H. rufipes males' body size showed high variation in urban habitats. This might be explained by the heterogeneous habitat structure in cities, because habitats with less or no street lights can even exist within cities (Rich & Longcore, 2006). Likewise, the body size in males of the diurnal H. affinis showed larger variability in the city in recent times (2016-2017) compared with the past . This finding might be also reasoned by higher habitat heterogeneity in the city, ranging from highly urbanised to almost natural areas, making Berlin to one of the greenest but simultaneously highly populated European cities (Schewenius et al., 2014).
The hypothetical explanation based on activity during the day and sex-dependent dispersal abilities would explain why both sexes of the diurnal H. affinis and the less mobile females of the nocturnal H. rufipes showed no changes in body size through time and that morphological trait changes might be dependent on the respective activity pattern of a species, and different behaviours of their sexes. In the diurnal H. affinis, sex-specific adaptation in coloration to urbanisation across the same timeframe was found (Keinath et al., 2020).

| Spatial nitrogen enrichments in carabids
We detected higher nitrogen isotopic enrichments in individuals of both species from landscapes with intensive agriculture compared to urban habitats. In particular, we found higher nitrogen isotopic signatures in all tissues and body components representing nutritive signatures of the larval stages (Gratton & Forbes, 2006;Peterson & Fry, 1987). Stable nitrogen enrichments might be higher in individuals because of their position in the food web, or when the respective environment is highly enriched by nitrogen, such as in agricultural landscapes due to fertiliser use (Birkhofer et al., 2011). Because both ground beetle species are feeding predominantly on weed seeds (Freyer & Aly, 1974;Jenkinson, 2001;Shearer et al., 1974), our results indicate that larvae occurring in agricultural habitats might have fed on highly nitrogenic enriched weed seeds, what might be caused by fertiliser use. This was also described by Birkhofer et al. (2011) for other arthropods, showing that nitrogen isotopic compositions are increasing with the intensity level of agricultural land usage.
However, in H. affinis' tissues, representing adult stage, we detected no difference in the stable nitrogen isotope composition between specimens from agricultural and urban habitats, suggesting that H.
affinis did not disperse into isotopically distinct habitats after larval development. This particularly also seems to apply to H. affinis adults from urban habitats seemingly living in habitats with nitrogen isotope ratios comparable to those of intense agricultural ones or feeding on different diets compared to rural beetles.
The assumption that some urban habitats might be similarly high enriched by nitrogen as agricultural ones, may again be underlined by the higher variability of nitrogen isotope values in H. rufipes tissues, representing adult stages, in urban habitats compared to agricultural ones. Agricultural habitats are more homogenous, in particular due to the presence of monocultures (Jongman, 2002), whereas urban environments exhibit more heterogeneous structures such as parks, private gardens, ruderal areas and green strips (Falk, 1976;Gill & Bonnett, 1973;Tischler, 1973). Thus, urban habitats are also variable in their nitrogen enrichments (Pyšek, 1995), depending on the degree of anthropogenic impact, that is the proximity of streets due to automobile exhaust (Baker et al., 2001), the use of fertiliser for maintaining lawns (Muchovej & Rechcigl, 1994), the 'structure' of the city (amount and pattern of green versus built cover space), and the deposit of excretions of pets (Zhu et al., 2004). Increased nitrogen isotopic variability was also found in the European Common Frog, Rana temporaria, when occurring in non-agricultural environments in comparison with agricultural landscapes within the Berlin-Brandenburg region (Niemeier et al., 2020).

| Spatial carbon enrichments in carabids
We found no significant differences in carbon signatures in both species' tissues and body components between agricultural and urban habitats. However, in H. rufipes higher carbon isotope signatures were visible when sampled from agricultural habitats. Although this effect was not significant, it might be explained by the higher proportion of C4 plants, like corn, in arable fields than in the city (Degens, 1969;Schwarcz, 1969).

| CON CLUS IONS
Our results revealed spatio-temporal intraspecific morphological and biochemical trait changes in response to human-induced environmental alterations. Morphological changes occurred across the relative short timeframe of 125 years in our study area, depend on the respective species, their sex and activity pattern. Furthermore, we found nitrogen enrichments in the environment to influence biochemical traits in specimens, occurring in habitats that are differently human-induced.

CO N FLI C T O F I NTER E S T S TATEM ENT
We declare no conflicts of interest.  Note: Results of Wilcoxon rank sum tests for comparisons between tissues of females and males and between beetles originated from agricultural and urban habitats. Significant differences are given in bold.

TA B L E A 3
Stable 14 N and 13 C isotope enrichments (in %) in cuticula, legs, and muscles of Harpalus affinis and H. rufipes between sexes and specimens from agricultural and urban habitats.
TA B L E A 4 Stable 14 N and 13 C isotope enrichments between cuticula, legs, and muscles of Harpalus affinis and H. rufipes from agricultural and urban habitats. TA B L E A 5 Stable 15 N and 13 C isotope enrichments (in ‰) in cuticula, legs, and muscles of Harpalus affinis and H. rufipes from agricultural and urban habitats.